CN114573562A - Compound containing nicotinic acid and triazole as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a nicotinic acid-containing bistriazole compound as well as a preparation method and application thereof. The compound can be used for preparing anti-tumor (chronic granulocytic leukemia) medicines, and has certain Bcr-Abl inhibition effect^WT、Bcr‑Abl^T315IKinase activity and certain cell proliferation inhibiting activity on K562 cells. The introduction of an L-proline structure can expand the structural diversity of a Bcr-Abl kinase inhibitor, and an activity test shows that the nicotinic acid triazole has an important effect on the inhibitory activity of a compound, can improve the affinity between a receptor and the compound, and can be used as a Bcr-Abl tyrosine kinaseA pharmaceutically effective fragment of an enzyme inhibitor.

Description

Compound containing nicotinic acid bitriazole and preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic compound preparation, and relates to a nicotinic acid contained triazole compound, and a preparation method and application thereof.

Background

Chronic Myeloid Leukemia (CML) is a malignant clonal proliferative disease occurring in myeloid hematopoietic stem cells, accounting for up to 15% -20% of adult leukemia patients, and is characterized by the ability to detect Philadelphia chromosome (Ph chromosome) in CML patients. The Ph chromosome is a breakpoint aggregation cluster-Elbeson (Bcr-Abl) fusion gene formed by the mutual translocation of the normal human chromosome 22 and the normal human chromosome 9, and the fusion gene codes for generating a Bcr-Abl fusion protein with continuous activation of tyrosine kinase activity. Small molecule tyrosine kinase inhibitors aiming at Bcr-Abl as targets are marketed in the market at present, but the small molecule tyrosine kinase inhibitors have the problems of drug resistance, other clinical adverse reactions and the like. Accordingly, research and development of novel Bcr-Abl tyrosine kinase inhibitors have become one of hot spots in the pharmaceutical field.

Disclosure of Invention

The invention aims to provide a compound containing nicotinic acid bitriazole and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a compound containing nicotinic acid bitriazole is disclosed, and the structural formula of the compound is as follows:

wherein R' is

The preparation method of the nicotinic acid bitriazole compound comprises the following steps:

1) carrying out substitution reaction on L-hydroxyproline and di-tert-butyl dicarbonate in an ice-water bath to prepare L-hydroxyproline protected by tert-butoxycarbonyl;

2) condensing the tert-butoxycarbonyl protected L-hydroxyproline with 5-amino-2-chlorotrifluoromethylbenzene to produce (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester;

3) reacting (2S,4R) -tert-butyl 2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylate with methanesulfonyl chloride to give tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate;

4) under the protection of nitrogen, carrying out substitution reaction on tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methylsulfonyloxypyrrole-1-carboxylate and sodium azide to generate tert-butyl (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylate;

5) under the action of sodium ascorbate and copper sulfate pentahydrate, condensing a 5-ethynylnicotinamide compound and (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester to obtain a pyridine-triazole compound protected by a Boc protecting group;

6) reacting trifluoroacetic acid with a pyridine triazole compound protected by a Boc protecting group in an anhydrous dichloromethane solvent, and removing the protecting group to obtain the compound containing the pyridine triazole compound.

In a further improvement of the invention, the 5-ethynyl nicotinamide compound is prepared by the following process:

a) reacting 5-bromo-nicotinic acid and thionyl chloride with an amine compound to prepare ammoniated 5-bromo-nicotinic acid;

b) under the protection of nitrogen, ammoniated 5-bromonicotinic acid reacts with trimethylsilyl acetylene to prepare 5- (2-trimethylsilyl) ethynyl nicotinamide compound;

c) removing trimethylsilyl group from 5- (2-trimethylsilyl) ethynyl nicotinamide compound by tetrabutylammonium fluoride trihydrate to obtain the 5-ethynyl nicotinamide compound.

The further improvement of the invention is that the specific process of the step a) is as follows: under nitrogen, dropwise adding thionyl chloride into 5-bromonicotinic acid at 25-30 ℃, performing reflux reaction to obtain crude acid chloride, dissolving the crude acid chloride into dichloromethane, adding the dichloromethane solution of an amine compound at 0 ℃, and stirring to obtain ammoniated 5-bromonicotinic acid;

the specific process of the step b) is as follows: adding ammoniated 5-bromonicotinic acid, copper iodide and tetrakis (triphenylphosphine) palladium into a reaction vessel, then sealing the vessel by using a rubber plug, vacuumizing and backfilling nitrogen, adding trimethylsilyl acetylene after adding triethylamine, and carrying out heating reflux reaction to obtain a 5- (2-trimethylsilyl) ethynyl nicotinamide compound;

the specific process of the step c) is as follows: adding tetrabutylammonium fluoride trihydrate into tetrahydrofuran solution dissolved with 5- (2-trimethylsilyl) ethynyl nicotinamide compound, and stirring at 25-30 ℃ to obtain the 5-ethynyl nicotinamide compound.

The further improvement of the invention is that the amine compound is morpholine, cyclopropylamine, N-diethylamine or N, N-dimethyl-1, 2-ethylenediamine;

the further improvement of the invention is that the specific process of the step 1) is as follows: dissolving L-hydroxyproline in tetrahydrofuran and sodium hydroxide solution at 0 ℃, adding di-tert-butyl dicarbonate in an ice-water bath, reacting at 25-30 ℃, concentrating under the condition of continuous ice-water bath, and performing substitution reaction on a concentrated product in the ice-water bath to prepare the tert-butoxycarbonyl protected L-hydroxyproline.

The further improvement of the invention is that the specific process of the step 2) is as follows: dissolving L-hydroxyproline protected by tert-butoxycarbonyl in dichloromethane, adding triethylamine, cooling to 0 ℃, dropwise adding a dichloromethane solution containing ethyl formate, reacting for 30-60 min to generate an active intermediate, then dropwise adding a 5-amino-2-chlorotrifluoromethylbenzene solution into the generated active intermediate at 0 ℃, and continuously stirring at 25-30 ℃ to obtain a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester.

The further improvement of the invention is that the specific process of the step 3) is as follows: dissolving a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester in dichloromethane, cooling to 0 ℃, adding triethylamine, stirring for 15min, dropwise adding methanesulfonyl chloride, and reacting at 25-30 ℃ to obtain (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylic acid tert-butyl ester;

the specific process of the step 4) comprises the following steps: dissolving a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methylsulfonyloxypyrrole-1-carboxylic acid tert-butyl ester in DMF, adding sodium azide, reacting under the protection of nitrogen at 65-70 ℃, cooling to 25-30 ℃, and precipitating a white solid in ice water to obtain (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester.

The further improvement of the invention is that the specific process of the step 5) is as follows: dissolving a compound 5-ethynylnicotinamide and a compound (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester in a mixed solvent of ethanol and water, and then adding sodium ascorbate and copper sulfate pentahydrate to obtain a suspension; stirring and concentrating the suspension to obtain a pyridine-bis-triazole compound protected by a Boc protecting group;

the specific process of the step 6) is as follows: dissolving the pyridine-triazole compound protected by the Boc protecting group in 30mL of anhydrous dichloromethane, dropwise adding trifluoroacetic acid at 0 ℃, and stirring at 25-30 ℃ to obtain the pyridine-triazole compound.

The application of the nicotinic acid contained bitriazole compound in preparation of Bcr-Abl kinase inhibitors.

The invention is further improved in that the Bcr-Abl kinase is a wild-Abl kinase or a T315I mutant Abl kinase.

The application of the nicotinic acid bitriazole compound in preparation of antitumor drugs.

The invention is further improved in that the anti-tumor drug is a drug for resisting leukemia cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention utilizes the reaction of the ring-pulling head, the click chemistry, the acylation, the condensation and the like to synthesize the target compound and constructs a compound library, the compound is a Bcr-Abl small molecule tyrosine kinase inhibitor with a novel molecular structure, and the structure of the target compound is represented by means of MS, NMR and the like. The invention adopts a fragment-based drug design strategy, uses the nicotinic acid-triazole as a hinge region binding fragment, introduces L-proline as a flexible Linker to construct a small molecule compound library with kinase inhibitory activity, and discovers the tyrosine kinase inhibitor with Bcr-Abl kinase inhibitory activity by ADP-Glo kinase activity screening. Kinase screening tests show that the compounds have certain inhibitory activity on both Abl kinase and T315I mutant Abl kinase, wherein the activity on Abl kinase is optimal when R is cyclopropylamino. Cell proliferation tests show that most compounds have certain inhibitory activity on K562 cells. Wherein the antiproliferative activity is best when R is cyclopropylamino. The analysis of the structure-activity relationship finds that: the derivative introduced with the L-proline has good spatial matching with the ATP site of the Abl kinase, and the action mode is consistent with that of a reference small molecule imatinib. The introduction of the acylated amino group on the carboxyl of the nicotinic acid triazole can improve the affinity of the micromolecule and the receptor, and can be used as a novel drug effect fragment for inhibiting tyrosine kinase by taking Bcr-Abl as a target.

Drawings

FIG. 1 is a synthetic route diagram of the present invention.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

The structural formula of the nicotinic acid bitriazole compound is as follows:

wherein R' is specifically as follows:

referring to fig. 1, the preparation method of the compound containing nicotinic acid bitriazole as described above is as follows:

1) reacting 5-bromo-nicotinic acid and thionyl chloride with an amine compound to prepare ammoniated 5-bromo-nicotinic acid; wherein the amine compound is morpholine, cyclopropylamine, N-diethylamine or N, N-dimethyl-1, 2-ethylenediamine;

2) under the protection of nitrogen, ammoniated 5-bromonicotinic acid reacts with trimethylsilyl acetylene to prepare 5- (2-trimethylsilyl) ethynyl nicotinamide compound;

3) removing trimethylsilyl from 5- (2-trimethylsilyl) ethynyl nicotinamide compound by tetrabutylammonium fluoride trihydrate to obtain 5-ethynyl nicotinamide compound;

4) carrying out substitution reaction on L-hydroxyproline and di-tert-butyl dicarbonate in an ice-water bath to prepare L-hydroxyproline (Boc-L-hydroxyproline) protected by tert-butoxycarbonyl;

5) condensation of Boc-L-hydroxyproline with 5-amino-2-chlorotrifluoromethylbenzene to give (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester;

6) reacting (2S,4R) -tert-butyl 2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylate with methanesulfonyl chloride to give tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate;

7) under the protection of nitrogen, tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methylsulfonyloxypyrrole-1-carboxylate and sodium azide are subjected to substitution reaction to generate tert-butyl (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylate.

8) Under the action of sodium ascorbate and copper sulfate pentahydrate, 5-ethynylnicotinamide compound and (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester are condensed to obtain the pyridine-triazole compound protected by Boc protecting group.

9) Reacting trifluoroacetic acid with a pyridine-triazole compound protected by a Boc protecting group in an anhydrous dichloromethane solvent, and removing the protecting group to obtain the pyridine-triazole compound according to claim 1.

The specific process of the step 1) is as follows: thionyl chloride was added dropwise to solid 5-bromonicotinic acid at 25-30 ℃ under nitrogen, refluxed for 2h, and volatiles were removed in vacuo. The crude acid chloride is dissolved in dichloromethane, slowly added into dichloromethane solution containing amine compounds at 0 ℃, and stirred for 12 h. Aqueous potassium carbonate solution was added, and the aqueous layer was extracted with dichloromethane. The organic extract was dried over sodium sulfate, concentrated in vacuo, and the crude product was purified by column chromatography to give 5-bromonicotinic acid as a white solid, aminated.

The specific process of the step 2) is as follows: ammoniated 5-bromonicotinic acid, copper iodide, and tetrakis (triphenylphosphine) palladium were added to the reaction vessel. The container was then sealed with a rubber stopper, evacuated and backfilled with nitrogen. Triethylamine is used as a base and a solvent, and the injection is carried out by using an injector. And (3) after 5min at 25-30 ℃, adding trimethylsilyl acetylene, heating and refluxing for reaction for 12h, and recording the reaction completion by thin-layer chromatography. The reaction is cooled to 25-30 ℃, and quenched with 50mL of water. The solution was then diluted with ethyl acetate and filtered. The filtrate was washed with water until no blue color of the copper complex was visible in the aqueous phase. The combined aqueous layers were extracted with ethyl acetate and repeated three times. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. And purifying the crude product by flash column chromatography to obtain a product 5- (2-trimethylsilyl) ethynyl nicotinamide compound.

The specific process of the step 3) is as follows: adding tetrabutylammonium fluoride trihydrate into tetrahydrofuran solution dissolved with 5- (2-trimethylsilyl) ethynyl nicotinamide compound. And stirring the reaction solution at 25-30 ℃ for 12 h. The solution was then evaporated to remove tetrahydrofuran and the residue was dissolved in ethyl acetate. The organic phase was washed with water and brine, and dried over anhydrous sodium sulfate. Removing the solvent to obtain a crude product, and purifying by chromatography to obtain the product 5-ethynyl nicotinamide compound.

The specific process of the step 4) is as follows: l-hydroxyproline was dissolved in tetrahydrofuran and sodium hydroxide solution at 0 ℃. Di-tert-butyl dicarbonate is added to the ice-water bath. The mixture is reacted for 6 hours at 25-30 ℃ until complete reaction is detected by thin layer chromatography. The reaction solution was concentrated in vacuo, cooled in an ice-water bath, acidified with hydrochloric acid and diluted with ethyl acetate. The mixture was then extracted with ethyl acetate. The combined organic layers were washed with brine and then water was removed with anhydrous sodium sulfate. And concentrating to obtain colorless oily Boc-L-hydroxyproline which can be continuously used without purification.

The specific process of the step 5) is as follows: the tert-butoxycarbonyl protected L-hydroxyproline was dissolved in methylene chloride and triethylamine was added. The solution was cooled to 0 ℃ and a solution of ethyl chloroformate in dichloromethane was added dropwise. After reacting for 30 min-1 h, generating an active intermediate. Then, the 5-amino-2-chloro trifluoromethyl benzene solution is dripped into the solution at the temperature of 0 ℃, and the stirring is continued for 12 hours at the temperature of 25-30 ℃. The reaction solution was diluted with dichloromethane, washed twice with saturated sodium bicarbonate solution, twice with water and 1 time with brine. The organic phase is dried over anhydrous sodium sulfate, filtered and chromatographed to obtain the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester.

The specific process of the step 6) is as follows: the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester was dissolved in dichloromethane and cooled to 0 ℃. Triethylamine was added and stirred for 15 min. Dropwise adding methylsulfonyl chloride, and reacting for 12 hours at 25-30 ℃. The reaction was quenched with water and washed with brine. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate.

The specific process of the step 7) is as follows: the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylic acid tert-butyl ester was dissolved in DMF and sodium azide was added. The mixture reacts for 16 hours under the protection of nitrogen at 65-70 ℃. Then the reaction solution was cooled to 25-30 ℃ and a white solid precipitated in ice water. The reaction mixture was extracted 3 times with ethyl acetate. The organic phases were combined, washed 1 time with brine, dried over anhydrous sodium sulfate and purified by chromatography to give (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester.

The specific process of the step 8) is as follows: dissolving a compound 5-ethynylnicotinamide and a compound (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester in a mixed solvent of ethanol and water in equal proportion. Sodium ascorbate and copper sulfate pentahydrate were then added to obtain a suspension. The suspension was stirred at 30 ℃ for 24h, and the reaction was concentrated in vacuo and extracted with ethyl acetate. Drying with anhydrous sodium sulfate, filtering, and purifying by chromatography to obtain the pyridine-triazole compound protected by the Boc protecting group.

The specific process of the step 9) is as follows: dissolving the pyridine-triazole compound protected by the Boc protecting group in anhydrous dichloromethane, and dropwise adding trifluoroacetic acid at 0 ℃. The solution was stirred at 25-30 ℃ for 2h, diluted with dichloromethane, adjusted to pH 8 with sodium bicarbonate solution and washed 3 times with water. The organic layer was dried over sodium sulfate, filtered, and purified by chromatography on ethyl acetate to obtain a pyridinetriazole compound as claimed in claim 1.

An application of a nicotinic acid contained bitriazole compound in preparation of drugs for inhibiting Abl kinase and T315I mutant Abl kinase activity.

The further improvement of the invention lies in the application of the compound in preparing an inhibitor for resisting the wild-Abl kinase and the T315I mutant Abl kinase.

An application of a nicotinic acid contained bistriazole compound in preparation of antitumor drugs.

The following description will be made by way of specific examples.

Example 1

A compound containing nicotinic acid bitriazole, R is

The preparation method comprises the following steps:

1)4- [ (5-Bromopyridin-3-yl) carbonyl]Synthesis of morpholine: thionyl chloride (36mL, 494mmol) was added dropwise to solid 5-bromonicotinic acid (5.00g, 24.7mmol) at room temperature (25-30 ℃ C.) under nitrogen. The resulting mixture was refluxed for 2h and the volatiles were removed in vacuo. Crude acid chlorineThe compound was dissolved in anhydrous dichloromethane (30mL) and the solution was added slowly to a solution of morpholine (4.74mL, 54.4mmol) in dichloromethane (30mL) at 0 ℃. Stirring was continued overnight. 20mL of a 2mol/L aqueous potassium carbonate solution was added, and the aqueous layer was extracted three times with 15mL of methylene chloride each time. The organic extract was dried over anhydrous sodium sulfate, concentrated in vacuo and the crude product was purified by chromatography on silica gel (vol.% petroleum ether: ethyl acetate: 1) to give 5.55g of a white solid in 83% yield. Mp 76-78 ℃; EI-MS (M/z)271[ M]⁺.

2)4- [ (5- ((2-trimethylsilyl) ethynyl) pyridin-3-yl) carbonyl]Synthesis of morpholine: reacting 4- [ (5-bromopyridin-3-yl) carbonyl]Morpholine (2.03g,7.5mmol), copper iodide (10%, 0.31g,1.61mmol), and tetrakis (triphenylphosphine) palladium (10%, 1.86g, 1.61mmol) were added to a 100mL two-necked round bottom flask with a condenser and magnetic stirring. The container was then sealed with a rubber stopper, evacuated and backfilled with nitrogen (3 times). Triethylamine (30mL) was used as the base and solvent and injected with a syringe. And (3) after 5min at 25-30 ℃, adding trimethylsilyl acetylene (4.74mL,48.36mmol), heating and refluxing for reaction for 12h, and recording the complete reaction by thin-layer chromatography. The reaction is cooled to 25-30 ℃, and quenched with 50mL of water. The solution was then diluted with 50mL of ethyl acetate and filtered. The filtrate was washed with water until no blue color of the copper complex was visible in the aqueous phase. The combined aqueous layers were extracted with 30mL of ethyl acetate and repeated three times. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash column chromatography (vol.% petrol ether: ethyl acetate: 5: 1) to give 1.2g of product in 57% yield. EI-MS (M/z)288[ M]⁺。

3)4- [ (5-ethynylpyridin-3-yl) carbonyl]Synthesis of morpholine: in solution with 4- [ (5- ((2-trimethylsilane) ethynyl) pyridin-3-yl) carbonyl]To a 50mL tetrahydrofuran solution of morpholine (1.2g,4.3mmol) was added a tetrahydrofuran solution containing tetrabutylammonium fluoride trihydrate (1M,15 mL-15 mmol). And stirring the reaction solution at 25-30 ℃ for 12 h. The solution was then evaporated to remove tetrahydrofuran and the residue was dissolved in ethyl acetate (50 mL). The organic phase was washed twice with 20mL portions of water and once with 30mL of brine and dried over anhydrous sodium sulfate. Removing solvent to obtain crude product, and performing chromatography (volume ratio petroleum ether): ethyl acetate 1: 1) purification gave 0.62g of compound in 66% yield. EI-MS (M/z)215[ M-H]^-.

4) Preparation of Boc-L-hydroxyproline: l-hydroxyproline (9.0g, 68.7mmol) was dissolved in tetrahydrofuran (82.5mL) and sodium hydroxide solution (1M, 82.5mL) at 0 ℃. Di-tert-butyl dicarbonate (15.75mL, 68.7mmol) was added to the ice-water bath. The mixture is reacted for 6 hours at 25-30 ℃ until complete reaction is detected by thin layer chromatography. The reaction solution was concentrated in vacuo to about 80mL, cooled in an ice-water bath, acidified to pH 2-3 with 1M hydrochloric acid, and diluted with 100mL ethyl acetate. Extract 3 times with 50mL of ethyl acetate. The combined organic layers were washed 2 times with 50mL of brine and then dried over anhydrous sodium sulfate to give a colorless oil that was used without further purification.

5) Preparation of tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylate: Boc-L-hydroxyproline (3.48g, 15.31mmol) was dissolved in 50mL of dichloromethane and triethylamine (1.5mL, 15.31mmol) was added. The solution was cooled to 0 deg.C and a solution of ethyl chloroformate (2mL, 15.31mmol) in 2mL of methylene chloride was added dropwise. After reacting for 30 min-1 h, generating an active intermediate. Then, 10mL of a solution of 5-amino-2-chlorotrifluoromethylbenzene (2.7g, 13.92mmol in dichloromethane) was added dropwise to the solution at 0 ℃ and stirring was continued at 25-30 ℃ for 12 hours. The reaction solution was diluted with 50mL of dichloromethane, washed 2 times with 30mL saturated sodium bicarbonate solution, twice with 30mL water, and 1 time with 30mL brine. The organic phase was dried over anhydrous sodium sulfate, filtered and chromatographed (volume ratio petroleum ether: ethyl acetate: 3: 1) to give 4.26g of product in 75% yield.

6) Preparation of (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylic acid tert-butyl ester: the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester (3.79g, 9.27mmol) was dissolved in 80mL dichloromethane and cooled to 0 ℃. Triethylamine (1.55mL, 11.12mmol) was added and stirred for 15 min. Dropwise adding methylsulfonyl chloride (0.86mL, 11.12mmol), and reacting at 25-30 ℃ for 12 h. The reaction was quenched with 20mL of water and washed with 30mL of brineOnce. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give the product 3.87g, 86% yield. EI-MS (M/z)487.05.10[ M + H]⁺,485.00[M-H]^-.。

7) Preparation of tert-butyl (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylate: the compound (2S,4R) -tert-butyl 2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate (3.78 g, 7.76mmol) was dissolved in 10mL of DMF, and sodium azide (0.99g, 15.52mmol) was added. The mixture is reacted for 16 hours under the protection of nitrogen at 65-70 ℃. Then the reaction solution was cooled to 25-30 ℃, poured into 100mL of ice water, and a white solid precipitated. The reaction mixture was extracted 3 times with 80mL portions of ethyl acetate. The organic phases were combined, washed 1 time with 50mL of brine, dried over anhydrous sodium sulfate and purified by chromatography (vol.% petroleum ether: ethyl acetate: 5: 1) to give 2.69g of product in 80% yield. Mp 57.6-58.2 ℃; EI-MS (M/z)434[ M + H]⁺,432[M-H]^-.

8) Preparation of (2S,4S) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4- (4- (5- (morpholine-4-carbonyl) pyridin-3-yl) -1H-1,2, 3-triazol-1-yl) pyrrole-1-carboxylic acid tert-butyl ester: a100 mL round bottom flask was charged with a mixture of 40mL ethanol and 40mL water. Then adding compound 4- [ (5-ethynylpyridin-3-yl) carbonyl]Morpholine (0.43g, 2.0mmol) and the compound (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester (0.8g, 2 mmol). Sodium ascorbate (0.1g, 0.5mmol) and copper sulfate pentahydrate (0.055g, 0.25mmol) were then added. The suspension is stirred at 30 ℃ for 24h, the reaction is concentrated in vacuo to 40mL and extracted three times with 30mL portions of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and chromatographed (volume ratio petroleum ether: ethyl acetate: 1) to give 0.97g of the product in 75% yield. EI-MS (M/z)650.25[ M + H]⁺,648.30[M-H]^-.

9) Preparation of (2S,4S) -N- (4-chloro-3- (trifluoromethyl) phenyl) -4- (4- (5- (morpholine-4-carbonyl) pyridin-3-yl) -1H-1,2, 3-triazol-1-yl) pyrrolidine-2-carboxamide: the compound (2S,4S) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4- (4- (5- (morpholine-4-carbonyl) pyri-dineTert-butyl pyridin-3-yl) -1H-1,2, 3-triazol-1-yl) pyrrole-1-carboxylate (0.53g, 0.81mmol) was dissolved in 30mL of anhydrous dichloromethane. At 0 deg.C, 2mL of trifluoroacetic acid was added dropwise. The solution was stirred at 25-30 ℃ for 2h, diluted with 50mL of dichloromethane, adjusted to pH 8 with sodium bicarbonate solution and washed 3 times with 30mL of water each time. The organic layer was dried over anhydrous sodium sulfate, filtered and purified by ethyl acetate chromatography to give the product NAZ10.37g, 84% yield. Mp is 79.5-83.7 ℃; EI-MS (M/z)550.10[ M + H]⁺,548.10[M-H]^-HRMS m/z vs C₂₄H₂₄ClF₃N₇O₃([M+H]⁺) The calculated value was 550.15812, found 550.15869.¹H NMR(400MHz,DMSO-d₆)δ10.37(s,1H),9.06(d,J＝ 1.8Hz,1H),8.95(s,1H),8.56(d,J＝1.7Hz,1H),8.23(d,J＝2.1Hz,1H),8.19(s,1H),7.95(d,J ＝8.8Hz,1H),7.61(d,J＝8.8Hz,1H),5.23–5.13(m,1H),4.01–3.93(m,1H),3.67–3.57(m, 8H),3.53–3.49(m,2H),2.89–2.81(m,1H),2.47–2.40(m,1H)。

The procedures for the synthesis of NAZ2, NAZ3 and NAZ4 were the same as those for the preparation of NAZ1 in example 1.

The compound NAZ2 takes cyclopropylamine (3.77mL) as a starting material, and the compound is obtained as a white solid in a yield of 63% through the steps 1) to 9); mp 192.1-193.2 ℃; EI-MS (M/z)520.20[ M + H]⁺,518.20[M-H]^-HRMS m/z vs C₂₃H₂₂ClF₃N₇O₂([M+H]⁺) The calculated value was 520.14756 and the experimental value was 520.14756.¹H NMR(400MHz, DMSO-d₆)δ10.37(s,1H),9.08(d,J＝2.1Hz,1H),8.94(s,1H),8.90(d,J＝2.1Hz,1H),8.73(d,J ＝4.3Hz,1H),8.54(t,J＝2.1Hz,1H),8.22(d,J＝2.5Hz,1H),7.95(dd,J＝8.8,2.6Hz,1H),7.60 (d,J＝8.8Hz,1H),5.29–5.09(m,1H),4.01–3.97(m,1H),3.58(s,1H),3.54–3.50(m,1H), 3.43–3.36(m,1H),2.98–2.79(m,2H),2.49–2.41(m,1H),0.76–0.71(m,2H),0.64–0.58(m, 2H).

Compound NAZ3 was obtained in 69% yield as a white solid compound from N, N-diethylamine (5.60mL) via steps 1) to 9). Mp 143.6-146.8 ℃; EI-MS (M/z)536.20[ M + H]⁺,534.15[M-H]^-HRMS m/z vs C₂₄H₂₆ClF₃N₇O₂([M+H]⁺) The calculated value was 536.17886, found 536.18003.¹H NMR(400MHz, DMSO-d₆)δ10.36(s,1H),9.06(d,J＝2.1Hz,1H),8.96(s,1H),8.51(d,J＝2.0Hz,1H),8.23(d,J ＝2.5Hz,1H),8.13(t,J＝2.1Hz,1H),7.94(dd,J＝8.8,2.5Hz,1H),7.60(d,J＝8.8Hz,1H),5.24 –5.13(m,1H),4.00–3.96(m,1H),3.56–3.44(m,3H),3.41–3.36(m,1H),3.21(d,J＝7.3Hz, 2H),2.92–2.80(m,1H),2.48–2.41(m,1H),1.18(t,J＝7.2Hz,3H),1.06(d,J＝9.6Hz,3H).

The compound NAZ4 is prepared from N, N-dimethyl-1, 2-ethylenediamine (4.8mL) as a starting material through the steps 1) to 9) to obtain a white solid with the yield of 64 percent and the Mp of 67.1-75.5 ℃; EI-MS (M/z)551.15[ M + H]⁺,549.30[M-H]^-HRMS m/z vs C₂₄H₂₇ClF₃N₈O₂([M+H]⁺) The calculated value was 551.18976, found 551.19049.¹H NMR(400MHz, DMSO-d₆)δ10.52(s,1H),9.09(s,1H),8.94(d,J＝13.2Hz,2H),8.76(s,1H),8.59(s,1H),8.24 (s,1H),7.96(d,J＝8.9Hz,1H),7.60(d,J＝8.8Hz,1H),5.25–5.15(m,1H),4.01–3.98(m,1H), 3.54–3.49(m,1H),3.45–3.34(m,3H),2.89–2.81(m,1H),2.44–2.41(m,3H),2.19(s,6H).

The nicotinic acid bitriazole compounds with anti-tumor activity prepared by the method are screened for Bcr-Abl kinase inhibitory activity.

The determination method specifically comprises the following steps:

the kinases Bcr-Abl, Bcr-Abl (T315I) and the substrate Abltide were purchased from Signal-Chem, and selected from ADP-Glo from Promega^TMThe enzyme inhibitory activity of the target compound is detected by the Kinase Assays detection kit, and the operation method is carried out according to the kit instructions.

In the Abl experiment, buffer (2X) (Tris 80mM, MgCl) was used₂20mM, BSA 0.2mg/ml, DTT 2mM) ATP (1mM) was diluted 80-fold and prepared as a buffer (2X) solution of ATP (125. mu.M); then preparing a mixed solution of ATP (62.5 mu M) -Abltide (0.5 mu g/mu L) according to the volume of 1:1 for later use by using the ATP (125 mu M) solution and the Abltide solution; with buffer (1X) (Tris 40mM, MgCl)₂10mM, BSA 0.1mg/ml, DTT 1 mM)) the Abl kinase solution was diluted 100-fold and formulated as Abl(1 ng/. mu.L) in buffer (1X) for use.

In the Abl (T315I) experiment, the procedures for ATP-Abltide and Abl (T315I) were as above, except that the concentrations of ATP and Abl (T315I) were 12.5. mu.M and 2 ng/. mu.L, respectively.

Four target compounds were each formulated with buffer (1X) to 1.5X 10^-5，1.5×10^-6，1.5×10^-7，1.5×10^-8，1.5×10^-9， 1.5×10^-10Adding 2 mu L of ATP-Abltide mixed solution, 1 mu L of sample solution and 2 mu L of enzyme solution into each well of a 384-well plate in turn according to the mol/L concentration gradient sample solution; adding 3 mu L of buffer solution and 2 mu L of ATP-Abltide mixed solution into a blank hole; adding 2. mu.L of ATP-Abltide mixed solution, 1. mu.L of buffer solution and 2. mu.L of enzyme solution into each control well, and incubating at 30 ℃ for 60min after the addition is finished; adding 5 μ L of ADP-Glo reagent, and incubating at 25 deg.C for 40 min; finally, Kinase detection reagent was added and incubated at 25 ℃ for 30 min. Measuring the luminescence value of each hole by using a chemiluminescence module of a PerkinElmer multifunctional microplate reader, and calculating the inhibition rate and IC (integrated Circuit) of the compound on Abl₅₀。

The kinase inhibitory activity of the nicotinic acid bitriazole compound with the structural formula is shown in Table 1

TABLE 1 Bitsucotinamide compounds vs Bcr-Abl^WTAnd Bcr-Abl^T315IInhibitory Activity of IC₅₀(nM)

Compound NAZ1-NAZ4 on Bcr-Abl^WTAnd Bcr-Abl^T315IThe results of the kinase inhibitory activity of (3) are shown in Table 1. It can be seen that most of the compounds have better inhibitory activity against Bcr-Abl kinase, among which the most active is compound NAZ2, which is against Bcr-Abl^WTAnd Bcr-Abl^T315IIC of₅₀Values were 1.94nM and 31.55nM, respectively. In addition, the activity of the compound NAZ1-NAZ4 is obviously reduced along with the increase of the molecular weight and the volume of the side chain of the amide (NAZ 1)<NAZ4<NAZ3<NAZ 2). The activity results show that the different pairs of substituents are kinase of the compoundThe inhibitory activity had a significant effect.

The proliferation inhibitory activity of the nicotinic acid-bitriazole compounds on tumor cells was determined as follows.

The MTT method is adopted to determine the proliferation inhibition activity of the nicotinic acid bitriazole compounds on tumor cells.

The nicotinic acid bitriazole compound provided by the invention has an anti-tumor effect. Has effect in inhibiting proliferation activity of tumor cells in vitro and in human leukemia cells (K562 cells), and can be used for treating leukemia.

Diluting human leukemia cells (K562 cells) in growth exponential phase to 10 with RPMI1640 medium⁴Cell solutions of the order of one/mL were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. mu.L/well and 5% CO at 37 ℃ in a culture medium₂Culturing for 12 h;

20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 1.5X 10^{- 7}mol/L， 1.5×10^-6mol/L，1.5×10^-5mol/L，1.5×10^-4mol/L, setting 3 multiple wells for each concentration, setting 6 multiple wells for negative control, adding cells without compound and imatinib as positive control, continuing at 37 deg.C and 5% CO₂Culturing for 48 h;

mu.L of MTT (5mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO₂Culturing for 4 hr, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating IC of compound according to the inhibition rate by linear regression method₅₀A value;

the formula for calculating the cell inhibition rate is as follows:

inhibition%;

the detection result shows that the nicotinic acid bitriazole compound has different degrees of in vitro inhibition effects on the tumor cells compared with the negative control group, and the results are shown in table 2.

TABLE 2 inhibitory Activity of nicotinic acid-bitriazoles on K562 cells IC₅₀(μM)

The cell activity screening test shows that the compound NAZ1-NAZ4 has certain cell proliferation inhibiting activity on K562 cells, and the IC of the compound₅₀Values were at the micromolar level, ranging from 0.82. mu.M to 28.83. mu.M. Wherein the most active compound is NAZ2, IC₅₀The value was 0.82. mu.M. For the nicotinic acid bitriazole compound, the results of the cellular activity and the kinase activity are consistent, and different amide side chains are introduced to a pyridine ring, so that the effect on the tumor cell proliferation inhibition activity is achieved. The compound NAZ2 has good inhibitory activity on K562 cells, and can be further studied.

The compound can be used for preparing anti-tumor (chronic granulocytic leukemia) medicines, and has certain Bcr-Abl inhibition effect^WT、 Bcr-Abl^T315IKinase activity and certain cell proliferation inhibiting activity on K562 cells. The introduction of an L-proline structure can expand the structural diversity of the Bcr-Abl kinase inhibitor, and an activity test shows that the niacin triazole has an important effect on the inhibitory activity of the compound, can improve the affinity between a receptor and the compound, and can be used as a pharmacodynamic fragment of the Bcr-Abl tyrosine kinase inhibitor.

Claims (10)

1. A compound containing nicotinic acid bitriazole is characterized in that the structural formula of the compound is as follows:

wherein R' is

2. The method for preparing the bitriazole nicotinate compound according to claim 1, wherein the method comprises the following steps:

1) carrying out substitution reaction on L-hydroxyproline and di-tert-butyl dicarbonate in an ice-water bath to prepare L-hydroxyproline protected by tert-butoxycarbonyl;

2) condensing the tert-butoxycarbonyl protected L-hydroxyproline with 5-amino-2-chlorotrifluoromethylbenzene to produce (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester;

3) reacting (2S,4R) -tert-butyl 2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylate with methanesulfonyl chloride to give tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate;

4) under the protection of nitrogen, carrying out substitution reaction on tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methylsulfonyloxypyrrole-1-carboxylate and sodium azide to generate tert-butyl (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylate;

5) under the action of sodium ascorbate and copper sulfate pentahydrate, condensing a 5-ethynylnicotinamide compound and (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester to obtain a pyridine-triazole compound protected by a Boc protecting group;

6) reacting trifluoroacetic acid with a pyridine triazole compound protected by a Boc protecting group in an anhydrous dichloromethane solvent, and removing the protecting group to obtain the compound containing the pyridine triazole compound.

3. The method for preparing a bitriazole nicotinate compound according to claim 2, wherein the 5-ethynyl nicotinamide compound is prepared by:

a) 5-bromo-nicotinic acid, thionyl chloride and amine compounds react to prepare ammoniated 5-bromo-nicotinic acid;

b) under the protection of nitrogen, ammoniated 5-bromonicotinic acid reacts with trimethylsilyl acetylene to prepare 5- (2-trimethylsilyl) ethynyl nicotinamide compound;

c) removing trimethylsilyl group from 5- (2-trimethylsilyl) ethynyl nicotinamide compound by tetrabutylammonium fluoride trihydrate to obtain the 5-ethynyl nicotinamide compound.

4. The preparation method of the compound containing nicotinic acid bitriazole according to claim 3, wherein the specific process in step a) is as follows: under nitrogen, dropwise adding thionyl chloride into 5-bromonicotinic acid at 25-30 ℃, performing reflux reaction to obtain crude acid chloride, dissolving the crude acid chloride into dichloromethane, adding the dichloromethane solution of an amine compound at 0 ℃, and stirring to obtain ammoniated 5-bromonicotinic acid;

the specific process of the step b) is as follows: adding ammoniated 5-bromonicotinic acid, copper iodide and tetrakis (triphenylphosphine) palladium into a reaction vessel, then sealing the vessel by using a rubber plug, vacuumizing and backfilling nitrogen, adding trimethylsilyl acetylene after adding triethylamine, and carrying out heating reflux reaction to obtain a 5- (2-trimethylsilyl) ethynyl nicotinamide compound;

the specific process of the step c) is as follows: adding tetrabutylammonium fluoride trihydrate into tetrahydrofuran solution dissolved with 5- (2-trimethylsilyl) ethynyl nicotinamide compound, and stirring at 25-30 ℃ to obtain the 5-ethynyl nicotinamide compound.

5. The preparation method of the compound containing nicotinic acid-bitriazole according to claim 2, characterized in that the specific process in step 1) is as follows: dissolving L-hydroxyproline in tetrahydrofuran and sodium hydroxide solution at 0 ℃, adding di-tert-butyl dicarbonate in an ice water bath, reacting at 25-30 ℃, concentrating under the condition of continuous ice water bath, and performing substitution reaction on a concentrated product in the ice water bath to prepare the tert-butoxycarbonyl protected L-hydroxyproline.

6. The preparation method of the compound containing nicotinic acid-bitriazole according to claim 2, characterized in that the specific process in step 2) is as follows: dissolving tert-butoxycarbonyl protected L-hydroxyproline in dichloromethane, adding triethylamine, cooling to 0 ℃, dropwise adding a dichloromethane solution containing ethyl chloroformate, reacting for 30-60 min to generate an active intermediate, then dropwise adding a 5-amino-2-chlorotrifluoromethylbenzene solution into the generated active intermediate at 0 ℃, and continuously stirring at 25-30 ℃ to obtain a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester.

7. The preparation method of the compound containing nicotinic acid-bitriazole according to claim 2, characterized in that the specific process in step 3) is as follows: dissolving a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester in dichloromethane, cooling to 0 ℃, adding triethylamine, stirring for 15min, dropwise adding methylsulfonyl chloride, and reacting at 25-30 ℃ to obtain (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylic acid tert-butyl ester;

the specific process of the step 4) is as follows: dissolving a compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methylsulfonyloxypyrrole-1-carboxylic acid tert-butyl ester in DMF, adding sodium azide, reacting under the protection of nitrogen at 65-70 ℃, cooling to 25-30 ℃, and precipitating a white solid in ice water to obtain the (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester.

8. The preparation method of the compound containing nicotinic acid-bitriazole according to claim 2, characterized in that the specific process in step 5) is as follows: dissolving a compound 5-ethynylnicotinamide and a compound (2S,4R) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrole-1-carboxylic acid tert-butyl ester in a mixed solvent of ethanol and water, and then adding sodium ascorbate and copper sulfate pentahydrate to obtain a suspension; stirring and concentrating the suspension to obtain a pyridine-triazole compound protected by a Boc protecting group;

the specific process of the step 6) is as follows: dissolving the pyridine-triazole compound protected by the Boc protecting group in 30mL of anhydrous dichloromethane, dropwise adding trifluoroacetic acid at 0 ℃, and stirring at 25-30 ℃ to obtain the pyridine-triazole compound.

9. The use of the bitriazole nicotinate containing compound as defined in claim 1 for preparing a Bcr-Abl kinase inhibitor.

10. The use of the bitriazole nicotinate compound as defined in claim 1 in the preparation of an antitumor medicament.

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